14

th
Proceeding the 6 International Conference on Green Technology
Maulana Malik Ibrahim State Islamic University / Malang, 18-19 September 2015

Arifah Khusnuryani1, Zainatul Fuad1

1Department of Biology, Faculty of Science and technology, UIN Sunan Kalijaga, Yogyakarta,
Indonesia
Email: anikarifah@yahoo.com

ABSTRACT

This research was conducted to observe the activity of ethanol extract of Awar-awar (Ficus septica Burm f)
leaves as antibacterial against Staphylococcus aureus ATCC 29523 and Escherichia coli ATCC 35218 based
on the formation of clear zone and analysis of cell leak. The result showed that ethanol extract of awar-
awar leaves performed antibacterial activity against S. aureus ATCC 29523 and E. coli ATCC 35218. Based
on the clear zone diamter, that antibacerial activity was categorized as medium. The analysis of cell leak
using spectrophotometer at λ 260 nm and 280 nm did not indicate that ethanol extract of awar-awar
leaves can cause cell leak on S. aureus ATCC 29523 and E. coli ATCC 35218.

Keywords
antibacterial activity, awar-awar leaves, E. coli ATCC 35218, S. aureus ATCC 29523

INTRODUCTION inhibit bacterial growth. Awar-awar (Ficus

Most of infectious diseases were
caused by bacteria. Staphylococcus aureus
and Escherichia coli are important pathogen
which usualy resistant to some medicines.
That resistance lead to difficulties in
choosing appropriate antimicrobial for
therapy. As reported by Depkes (2008) that
many antibiotics have been used for curing
of infectious diseases, however infection
problem still continue. Volk and Wheeler
(1993) stated that antibiotic cure can cause
resistance of bacteria, then we need new
product which potential as antibacterial to
solve that infection problem.
Plants contain some active chemical
compounds whic are potential to kill or
septica Burm F) is one of potential plants
that can be used for medication. This plant
can be found in Java, Madura, and Sulawesi
(Steenis, 2005). Awar-awar was used as
traditional medication in Bali. Awar-awar
leaves can be used as medicine for skin
disease, appendix disease, bisul, snake bite,
and asthma (Didik et al, 2002). The roots
can be used to cure some stomachaches
such as dysentri and cholerae, also to cure
toxin and asthma (Segatri, 1995).
Awar-awar contains some chemical
compounds such as saponin, flavonoid, and
tanin, also alkaloid such as tilosrebrin
(hauptalkaloid), tiloforin, septisin, and
antofin. The leaves and roots contain
stigmasterol and β-sitosterol. The roots
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Proceeding the 6 International Conference on Green Technology
Maulana Malik Ibrahim State Islamic University / Malang, 18-19 September 2015

also contain sterol and polyphenol (Didik et using ethanol to extract the active
al, 2002). compounds of awar-awar leaves.

Some researches have been

Tabel 1. Clear zone diameter after the treatment of
conducted to assess active compound of
plants or natural compounds to inhibit S. ethanol extract of Awar-awar leaves on S.
aureus and E. coli. Those researches are aureus ATCC 29523 and E. coli ATCC 35218
Azadirachta indica (mimba) seed to inhibit S. Diameter of clear
aureus (Ambarwati, 2007) and Anredera zone (mm)
cordifolia (binahong) leaves to inhibit S. No Treatment
aureus and Pseudomonas aeruginosa S. aureus E. coli
ATCC ATCC
(Khunaifi, 2010). Other investigations of S.
29523 35218
aureus growth inhibition were done using
Alpinia galanga (Yuharmen et al, 2002), Extract of Awar-awar
1 10% 7,7 8
Piper betle leaves (Poeloengan et al, 2006; leaves
Hermawan et al, 2007), Plumbago zeylanica 20% 9,7 9
L leaves (Poeloengan, 2009), and red betel
leaves (Reveny, 2011). While Curcuma 30% 8.0 8.3
xanthorriza (Meilisa, 2008), Artocarpus
altilis leaves (Sulistyaningsih et al, 2009), MATERIALS AND METHODS
and Morinda citrifolia (Dewi, 2010) were
used to inhibit E. coli. 1. Ethanol extraction of awar-awar
leaves powder
Based on the above explanation,
Extraction was proceed by maseration.
awar-awar has not been used for the Awar-awar leaves powder (200 g) was
investigation of S. aureus and E. coli dissolved with 800 mL ethanol 96%.
inhibition. Some researches using awar- Sample was homogenized using shaker
awar were the usage of methanol extract of at 120 rpm for 24 hours. Than, sample
awar-awar roots to inhibit Vibrio cholera was filtrated and the filtrat was
and E. coli (Sukadana, 2000), also ethanol evaporated until gel crude extract was
formed.
extract of awar-awar leaves againts
citotoxic activity of cancer cells (Sekti et al,
2010). 2. Antibacterial activity test of awar-
awar leaves extract using disc
Our preliminary test showed that difusion method
awar-awar leaves juice could inhibit S. Bacterial culture of S. aureus ATCC
aureus growth at concentration 25% (clear 29523 dan E. coli ATCC 35218 (0,1 mL
zone diameter 0,67 mm), 50% (clear zone each) from NB medium was separately
diameter 1,0 mm), and 75% (clear zone inoculated in NA medium using spread
diameter 2,0 mm). Those results show that plate method. Various ethanol extract
of awar-awar leaves (10%, 20%, 30%,
awar-awar leaves juice could inhibit S.
40%, dan 50%) were impregnated
aureus growth even the inhibition was onto 5 mm paper disk and then placed
weak. Therefore we conducted this research on the seeded NA medium using a
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Proceeding the 6 International Conference on Green Technology
Maulana Malik Ibrahim State Islamic University / Malang, 18-19 September 2015

sterile forceps. Plates were incubated at λ 260 nm for measurement of nucleic

for 24 hours at 37˚C. and the diameter acid nitrogen and at λ 280 nm for nitrogen
of the zone of inhibition around the of protein. The result is shown in Table 2.
disk is measured to the nearest
millimeter (Greenwood, 1995). Based
on Davis and Stout (1971), the
inhibition activity is categorized as
weak inhibition if the diameter of clear
zone is ≤ 5 mm, as medium inhibition if
the diameter is around 5-10 mm, as DISCUSSION
strong inhibition if the diameter
around 10-19 mm, and as very strong if
the diameter is ≥ 20 mm. Our preliminary study showed that
juice of awar-awar leaves, with aquades as
3. Analysis of cell leak (Bunduki et al, the solvent, can inhibit S. aureus ATCC
1995) 29523 growth with 2 mm clear zone
Bacterial suspension (10 mL) which diamater. Based on Davis and Stout (1971),
has been grown along 24 hours was that inhibition activity was categorized as
centrifuged at 3.500 rpm for 20 weak, because the clear zone was < 5 mm.
minutes. Filtrat was discharged and 5 However, we conclude that awar-awar
mL NaCl 0,85% was added into cell leaves were potential as antibacteria and
sediment. Then, extract awar-awar can be improved by extracting it using
leaves which produced smallest clear another solvent.
zone ( concentration 10%) was added
and incubated at 120 rpm, 37˚C, for 24
hours. Similar bacterial suspension
without extract addition was used as
comparison. Next, bacteria-extract
suspension wass centrifuged at 3.500
rpm for 20 minutes. The optical density
of supernatan then was analyzed
susing spectrophotometer UV-Vis at λ
260 nm for nucleic acid analysisi and at
λ 280 nm for protein analysis.

RESULTS
Data in Table 1 shows that ethanol
extract of awar-awar leaves with various
A B
concentartion can inhibit the growth of S.
aureus ATCC 29523 and E. coli ATCC 35218. Figure 1. The image of clear zone after the treatment of
Based on Davis and Stout (1971), the 20% (A) and 30% (B) ethanol extract of Awar-
inhibition was categorized as medium. The awar leaves on S. aureus ATCC 29523
images of clear zone are shown on Fig. 1
and 2.
Test of bacterial cell leak of S. aureus
ATCC 29523 and E. coli ATCC 35218 was
conducted using spectrophotometer UV-Vis
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Proceeding the 6 International Conference on Green Technology
Maulana Malik Ibrahim State Islamic University / Malang, 18-19 September 2015
Tabel 2. The result of cell leak analysis using
spectrophotometer at λ 260 nm and 280
Absorbance
Sample
λ 260 nm λ 280 nm
S. aureus ATCC 29523 1,035 0,463
E. coli ATCC 35218 0,885 0,434
S. aureus ATCC 29523 +
undetected undetected
extract
E. coli ATCC 35218 + extract undetected undetected
A B
Figure 2. The image of clear zone after the treatment research we found that the celar zone
of 20% (A) and 30% (B) ethanol extract of Awar-awar diameter was decrease when the extract
leaves on E. coli ATCC 35218
Ethanol was choosen as extraction
solvent, because it is a polar solvent, while
active compounds in awar-awar leaves are
polar compounds. Therefore it can extracte
the active compounds effectively. Moreover,
it is easy to get ethanol, ethanol is cheap,
not easy to evaporate, and not affect the
active compounds (Ahmad, 2006).
In this research we used S. aureus
ATCC 29523 (Gram positive) and E. coli
ATCC 35218 (Gram negative) to check the
antibacterial spectrum of awar-awar leaves.
ar-awar. Pelczar and Chan (1998) stated
that a compound has wide spectrum if that
compound can inhibit the growth of Gram
positive and Gram negative bacteria. If the
compound only inhibit the growth of Gram
positif or Gram negative bacteria, then it
can be categorized as a narrow spectrum
compound. Based on the result (Table 1
and Fig. 2), it can be concluded that ethanol
extract of awar-awar leaves has wide
spectrum antibacterial compound.
Awar-awar leaves contain some
secondary metabolites, such as flavonoid,
saponin, and tanin. Those secondary
metabolites can be extracted using ethanol
as solvent. Flavonoid can form complex
compoud with extracelullar protein. That
complex compound is able to be dissolved,
then can damage bacterial cell membrane
and followed by the release of intracellular
compounds of bacterial cell. Tanin can
wrinkle bacterial cell wall or membrane,
therefore it can disturb cell permeability
which lead to growth disturbation. While
saponin can damage cell membrane by
decreasing cell wall turgor which can lead
to cell wall lysis.
Usually, the diameter of clear zone
will increase parallel with the increase of
ekxtract concentration. However, in this
concentration was increased to 30%. Dewi
(2010) stated that the increase of clear zone
diameter not always corespond with the
increase of antibacterial concentration.
Antibacterial activity can be affected by the
concentration of antibacterial compound,
temperature, pH, microbial species, and
organic compound.
The result showed that S. aureus
ATCC 29523 and E. coli ATCC 35218 gave
different respond to our treatment. S.
aureus ATCC 29523 was more sensitive to
the extract than E. coli ATCC 35218. Cell
wall stucture of S. aureus ATCC 29523 is
more simple than of E. coli ATCC 35218.
The cell wall of S. aureus ATCC 29523 is
composed of one thick layer, while cell wall
of E. coli ATCC 35218 is composed of some
layers. Therefore, S. aureus ATCC 29523
more sensitive to antibacterial compounds.
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Proceeding the 6 International Conference on Green Technology
Maulana Malik Ibrahim State Islamic University / Malang, 18-19 September 2015
The bacterial respond to ethanol
extract of awar-awar leaves was also
observed by analysing the disturbance or
damage of membrane cell, based on the
bacterial growth medium turbidity.
Bacterial cell membrane damage was
measured based on the compounds which is
released by the cell, those are nitrogen from
nucleic acid (measured at λ 260 nm) and
nitrogen from protein (measured at λ 280
nm).
Data in Table 2 shows that the
absorbance at λ 260 nm and 280 nm were
undetected. That undectected absorbance
perhaps was caused by the intense color of
awar-awar leaves extract. Therefore
spectrophotometer UV-Vis could not detect
the nucleic acid and protein which were
released from bacterial cell.
Different result was shown by
Febriyanti (2010) who observed the effect
of betel leaf volatile oil fraction against
Gram positive bacteria such as Bacillus
subtilis, S. mutan, and S. aureus. While Azis
(2010) observed the antibacterial
mechanism of ethanol extract of white lily
bulbs againts Propionibacterium acnes, S.
aureus, dan S. epidermis. Both showed the
increase of absorbance when bacteria were
contacted with the extract. It can be
summarized that the different result of our
research with those two researches can be
affected by the different of the extracted
material, the kind of extract, and the strain
of bacteria. Next observation using other
methods is recommended to observe the
antibacterial mechanism of ethanol extract
of awar-awar leaves.
CONCLUSION
1. Ethanol extract of awar-awar leaves
can inhibit the growth of
Staphylococcus aureus ATCC 29523 and
Escherichia coli ATCC 35218. The
inhibition was categorized as medium
inhibition.
2. Antibacterial mechanism of ethanol
extract of awar-awar leaves against
Staphylococcus aureus ATCC 29523 and
Escherichia coli ATCC 35218 could not
be observed based on the analysis of
cell leak.
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Maulana Malik Ibrahim State Islamic University / Malang, 18-19 September 2015
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